SMART MODE:

NORMAL GENOMIC

• Dhingra N, Bruck I, Smith S, Ning B, Kaplan DL
• Dpb11 protein helps control assembly of the Cdc45.Mcm2-7.GINS replication fork helicase.
• J Biol Chem. 2015; 290: 7586-601
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• Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-phase cyclin-dependent kinase-phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS. We show here, using purified proteins from budding yeast, that Dpb11 alone binds to Mcm2-7 and that Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits the Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased replication protein A (RPA) interaction with origin DNA during S phase. We propose a model in which Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, although bound to Cdc45.Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7, and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45.Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase.

• Chen L, Madura K
• Yeast importin-alpha (Srp1) performs distinct roles in the import of nuclear proteins and in targeting proteasomes to the nucleus.
• J Biol Chem. 2014; 289: 32339-52
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• Srp1 (importin-alpha) can translocate proteins that contain a nuclear localization signal (NLS) into the nucleus. The loss of Srp1 is lethal, although several temperature-sensitive mutants have been described. Among these mutants, srp1-31 displays the characteristic nuclear import defect of importin-alpha mutants, whereas srp1-49 shows a defect in protein degradation. We characterized these and additional srp1 mutants to determine whether distinct mechanisms were required for intracellular proteolysis and the import of NLS-containing proteins. We determined that srp1 mutants that failed to import NLS-containing proteins (srp1-31 and srp1-55) successfully localized proteasomes to the nucleus. In contrast, srp1 mutants that did not target proteasomes to the nucleus (srp1-49 and srp1-E402Q) were able to import NLS-containing proteins. The proteasome targeting defect of specific srp1 mutants caused stabilization of nuclear substrates and overall accumulation of multiubiquitylated proteins. Co-expression of a member of each class of srp1 mutants corrected both the proteasome localization defect and the import of NLS-containing proteins. These findings indicate that the targeting of proteasomes to the nucleus occurs by a mechanism distinct from the Srp1-mediated import of nuclear proteins.

• Chen L, Madura K
• Degradation of specific nuclear proteins occurs in the cytoplasm in Saccharomyces cerevisiae.
• Genetics. 2014; 197: 193-7
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• The ubiquitin/proteasome system has been characterized extensively, although the site of nuclear substrate turnover has not been established definitively. We report here that two well-characterized nuclear proteins are stabilized in nuclear export mutants in Saccharomyces cerevisiae. The requirement for nuclear export defines a new regulatory step in intracellular proteolysis.

• Errico A, Aze A, Costanzo V
• Mta2 promotes Tipin-dependent maintenance of replication fork integrity.
• Cell Cycle. 2014; 13: 2120-8
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• Orderly progression of S phase requires the action of replisome-associated Tipin and Tim1 proteins, whose molecular function is poorly understood. Here, we show that Tipin deficiency leads to the accumulation of aberrant replication intermediates known as reversed forks. We identified Mta2, a subunit of the NuRD chromatin remodeler complex, as a novel Tipin binding partner and mediator of its function. Mta2 is required for Tipin-dependent Polymerase alpha binding to replicating chromatin, and this function is essential to prevent the accumulation of reversed forks. Given the role of the Mta2-NuRD complex in the maintenance of heterochromatin, which is usually associated with hard-to-replicate DNA sequences, we tested the role of Tipin in the replication of such regions. Using a novel assay we developed to monitor replication of specific genomic loci in Xenopus laevis egg extract we demonstrated that Tipin is directly required for efficient replication of vertebrate centromeric DNA. Overall these results suggest that Mta2 and Tipin cooperate to maintain replication fork integrity, especially on regions that are intrinsically difficult to duplicate.

• Wu L, Liu Y, Kong D
• Mechanism of chromosomal DNA replication initiation and replication fork stabilization in eukaryotes.
• Sci China Life Sci. 2014; 57: 482-7
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• Chromosomal DNA replication is one of the central biological events occurring inside cells. Due to its large size, the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time. Further, eukaryotic DNA replication is sophisticatedly regulated, and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle. The first step of replication initiation is the assembly of pre-replication complex (pre-RC). Since 1973, four proteins, Cdc6/Cdc18, MCM, ORC and Cdt1, have been extensively studied and proved to be pre-RC components. Recently, a novel pre-RC component called Sap1/Girdin was identified. Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells, respectively. At the transition of G1 to S phase, pre-RC is activated by the two kinases, cyclindependent kinase (CDK) and Dbf4-dependent kinase (DDK), and subsequently, RPA, primase-polalpha, PCNA, topoisomerase, Cdc45, poldelta, and polvarepsilon are recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication. As replication forks move along chromatin DNA, they frequently stall due to the presence of a great number of replication barriers on chromatin DNA, such as secondary DNA structures, protein/DNA complexes, DNA lesions, gene transcription. Stalled forks must require checkpoint regulation for their stabilization. Otherwise, stalled forks will collapse, which results in incomplete DNA replication and genomic instability. This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.

• Kang YH et al.
• Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase.
• Proc Natl Acad Sci U S A. 2013; 110: 19760-5
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• Chromosome transmission fidelity 4 (Ctf4) is a conserved protein required for DNA replication. In this report, interactions between human Ctf4 (hCtf4) and the replicative helicase containing the cell division cycle 45 (Cdc45)/minichromosome maintenance 2-7 (Mcm2-7)/Go, Ichi, Nii, and San (GINS) (CMG) proteins [human CMG (hCMG) complex] were examined. The hCtf4-CMG complex was isolated following in vitro interaction of purified proteins (hCtf4 plus the hCMG complex), coinfection of Spodoptera frugiperda (Sf9) insect cells with viruses expressing the hCMG complex and hCtf4, and from HeLa cell chromatin after benzonase and immunoprecipitation steps. The stability of the hCtf4-CMG complex depends upon interactions between hCtf4 and multiple components of the hCMG complex. The hCtf4-CMG complex, like the hCMG complex, contains DNA helicase activity that is more salt-resistant than the helicase activity of the hCMG complex. We demonstrate that the hCtf4-CMG complex contains a homodimeric hCtf4 and a monomeric hCMG complex and suggest that the homodimeric hCtf4 acts as a platform linking polymerase alpha to the hCMG complex. The role of the hCMG complex as the core of the replisome is also discussed.

• Thu YM, Bielinsky AK
• Enigmatic roles of Mcm10 in DNA replication.
• Trends Biochem Sci. 2013; 38: 184-94
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• Minichromosome maintenance protein 10 (Mcm10) is required for DNA replication in all eukaryotes. Although the exact contribution of Mcm10 to genome replication remains heavily debated, early reports suggested that it promotes DNA unwinding and origin firing. These ideas have been solidified by recent studies that propose a role for Mcm10 in helicase activation. Whereas the molecular underpinnings of this activation step have yet to be revealed, structural data on Mcm10 provide further insight into a possible mechanism of action. The essential role in DNA replication initiation is not mutually exclusive with additional functions that Mcm10 may have as part of the elongation machinery. Here, we review the recent findings regarding the role of Mcm10 in DNA replication and discuss existing controversies.

• Yeeles JT, Poli J, Marians KJ, Pasero P
• Rescuing stalled or damaged replication forks.
• Cold Spring Harb Perspect Biol. 2013; 5: 12815-12815
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• In recent years, an increasing number of studies have shown that prokaryotes and eukaryotes are armed with sophisticated mechanisms to restart stalled or collapsed replication forks. Although these processes are better understood in bacteria, major breakthroughs have also been made to explain how fork restart mechanisms operate in eukaryotic cells. In particular, repriming on the leading strand and fork regression are now established as critical for the maintenance and recovery of stalled forks in both systems. Despite the lack of conservation between the factors involved, these mechanisms are strikingly similar in eukaryotes and prokaryotes. However, they differ in that fork restart occurs in the context of chromatin in eukaryotes and is controlled by multiple regulatory pathways.

• Foltman M et al.
• Eukaryotic replisome components cooperate to process histones during chromosome replication.
• Cell Rep. 2013; 3: 892-904
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• DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription) complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.

• Tanaka S, Araki H
• Helicase activation and establishment of replication forks at chromosomal origins of replication.
• Cold Spring Harb Perspect Biol. 2013; 5: 10371-10371
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• Many replication proteins assemble on the pre-RC-formed replication origins and constitute the pre-initiation complex (pre-IC). This complex formation facilitates the conversion of Mcm2-7 in the pre-RC to an active DNA helicase, the Cdc45-Mcm-GINS (CMG) complex. Two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), work to complete the formation of the pre-IC. Each kinase is responsible for a distinct step of the process in yeast; Cdc45 associates with origins in a DDK-dependent manner, whereas the association of GINS with origins depends on CDK. These associations with origins also require specific initiation proteins: Sld3 for Cdc45; and Dpb11, Sld2, and Sld3 for GINS. Functional homologs of these proteins exist in metazoa, although pre-IC formation cannot be separated by requirement of DDK and CDK because of experimental limitations. Once the replicative helicase is activated, the origin DNA is unwound, and bidirectional replication forks are established.

• Kilkenny ML, De Piccoli G, Perera RL, Labib K, Pellegrini L
• A conserved motif in the C-terminal tail of DNA polymerase alpha tethers primase to the eukaryotic replisome.
• J Biol Chem. 2012; 287: 23740-7
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• The DNA polymerase alpha-primase complex forms an essential part of the eukaryotic replisome. The catalytic subunits of primase and pol alpha synthesize composite RNA-DNA primers that initiate the leading and lagging DNA strands at replication forks. The physical basis and physiological significance of tethering primase to the eukaryotic replisome via pol alpha remain poorly characterized. We have identified a short conserved motif at the extreme C terminus of pol alpha that is critical for interaction of the yeast ortholog pol1 with primase. We show that truncation of the C-terminal residues 1452-1468 of Pol1 abrogates the interaction with the primase, as does mutation to alanine of the invariant amino acid Phe(1463). Conversely, a pol1 peptide spanning the last 16 residues binds primase with high affinity, and the equivalent peptide from human Pol alpha binds primase in an analogous fashion. These in vitro data are mirrored by experiments in yeast cells, as primase does not interact in cell extracts with pol1 that either terminates at residue 1452 or has the F1463A mutation. The ability to disrupt the association between primase and pol alpha allowed us to assess the physiological significance of primase being tethered to the eukaryotic replisome in this way. We find that the F1463A mutation in Pol1 renders yeast cells dependent on the S phase checkpoint, whereas truncation of Pol1 at amino acid 1452 blocks yeast cell proliferation. These findings indicate that tethering of primase to the replisome by pol alpha is critical for the normal action of DNA replication forks in eukaryotic cells.

• Niimi A, Chambers AL, Downs JA, Lehmann AR
• A role for chromatin remodellers in replication of damaged DNA.
• Nucleic Acids Res. 2012; 40: 7393-403
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• In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells.

• Shen Z, Prasanth SG
• Emerging players in the initiation of eukaryotic DNA replication.
• Cell Div. 2012; 7: 22-22
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• Faithful duplication of the genome in eukaryotes requires ordered assembly of a multi-protein complex called the pre-replicative complex (pre-RC) prior to S phase; transition to the pre-initiation complex (pre-IC) at the beginning of DNA replication; coordinated progression of the replisome during S phase; and well-controlled regulation of replication licensing to prevent re-replication. These events are achieved by the formation of distinct protein complexes that form in a cell cycle-dependent manner. Several components of the pre-RC and pre-IC are highly conserved across all examined eukaryotic species. Many of these proteins, in addition to their bona fide roles in DNA replication are also required for other cell cycle events including heterochromatin organization, chromosome segregation and centrosome biology. As the complexity of the genome increases dramatically from yeast to human, additional proteins have been identified in higher eukaryotes that dictate replication initiation, progression and licensing. In this review, we discuss the newly discovered components and their roles in cell cycle progression.

• Bruck I, Kanter DM, Kaplan DL
• Enabling association of the GINS protein tetramer with the mini chromosome maintenance (Mcm)2-7 protein complex by phosphorylated Sld2 protein and single-stranded origin DNA.
• J Biol Chem. 2011; 286: 36414-26
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• The Cdc45-Mcm2-7-GINS (CMG) complex is the replication fork helicase in eukaryotes. Synthetic lethal with Dpb11-1 (Sld2) is required for the initiation of DNA replication, and the S phase cyclin-dependent kinase (S-CDK) phosphorylates Sld2 in vivo. We purified components of the replication initiation machinery and studied their interactions in vitro. We found that unphosphorylated or CDK-phosphorylated Sld2 binds to the mini chromosome maintenance (Mcm)2-7 complex with similar efficiency. Sld2 interaction with Mcm2-7 blocks the interaction between GINS and Mcm2-7. The interaction between CDK-phosphorylated Sld2 and Mcm2-7 is substantially inhibited by origin single-stranded DNA (ssDNA). Furthermore, origin ssDNA allows GINS to bind to Mcm2-7 in the presence of CDK-phosphorylated Sld2. However, unphosphorylated Sld2 blocks the interaction between GINS and Mcm2-7 even in the presence of origin ssDNA. We identified a mutant of Sld2 that does not bind to DNA. When this mutant is expressed in yeast cells, cell growth is severely inhibited with very slow progression into S phase. We propose a model wherein Sld2 blocks the interaction between GINS and Mcm2-7 in vivo. Once origin ssDNA is extruded from the Mcm2-7 ring and CDK phosphorylates Sld2, the origin ssDNA binds to CDK-phosphorylated Sld2. This event may allow the interaction between GINS and Mcm2-7 in vivo. Thus, CDK phosphorylation of Sld2 may be important to release Sld2 from Mcm2-7, thereby allowing GINS to bind Mcm2-7. Furthermore, origin ssDNA may stimulate the formation of the CMG complex by alleviating inhibitory interactions between Sld2 with Mcm2-7.

• Hartford SA, Luo Y, Southard TL, Min IM, Lis JT, Schimenti JC
• Minichromosome maintenance helicase paralog MCM9 is dispensible for DNA replication but functions in germ-line stem cells and tumor suppression.
• Proc Natl Acad Sci U S A. 2011; 108: 17702-7
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• Effective DNA replication is critical to the health and reproductive success of organisms. The six MCM2-7 proteins, which form the replicative helicase, are essential for high-fidelity replication of the genome. Many eukaryotes have a divergent paralog, MCM9, that was reported to be essential for loading MCM2-7 onto replication origins in the Xenopus oocyte extract system. To address the in vivo role of mammalian MCM9, we created and analyzed the phenotypes of mice with various mutations in Mcm9 and an intronic DNA replication-related gene Asf1a. Ablation of Mcm9 was compatible with cell proliferation and mouse viability, showing that it is nonessential for MCM2-7 loading or DNA replication. Mcm9 mutants underwent p53-independent embryonic germ-cell depletion in both sexes, with males also exhibiting defective spermatogonial stem-cell renewal. MCM9-deficient cells had elevated genomic instability and defective cell cycle reentry following replication stress, and mutant animals were prone to sex-specific cancers, most notably hepatocellular carcinoma in males. The phenotypes of mutant mice and cells suggest that MCM9 evolved a specialized but nonessential role in DNA replication or replication-linked quality-control mechanisms that are especially important for germ-line stem cells, and also for tumor suppression and genome maintenance in the soma.

• Kubota T, Hiraga S, Yamada K, Lamond AI, Donaldson AD
• Quantitative proteomic analysis of chromatin reveals that Ctf18 acts in the DNA replication checkpoint.
• Mol Cell Proteomics. 2011; 10: 110005561-110005561
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• Yeast cells lacking Ctf18, the major subunit of an alternative Replication Factor C complex, have multiple problems with genome stability. To understand the in vivo function of the Ctf18 complex, we analyzed chromatin composition in a ctf18Delta mutant using the quantitative proteomic technique of stable isotope labeling by amino acids in cell culture. Three hundred and seven of the 491 reported chromosomal proteins were quantitated. The most marked abnormalities occurred when cells were challenged with the replication inhibitor hydroxyurea. Compared with wild type, hydroxyurea-treated ctf18Delta cells exhibited increased chromatin association of replisome progression complex components including Cdc45, Ctf4, and GINS complex subunits, the polymerase processivity clamp PCNA and the single-stranded DNA-binding complex RPA. Chromatin composition abnormalities observed in ctf18Delta cells were very similar to those of an mrc1Delta mutant, which is defective in the activating the Rad53 checkpoint kinase in response to DNA replication stress. We found that ctf18Delta cells are also defective in Rad53 activation, revealing that the Ctf18 complex is required for engagement of the DNA replication checkpoint. Inappropriate initiation of replication at late origins, because of loss of the checkpoint, probably causes the elevated level of chromatin-bound replisome proteins in the ctf18Delta mutant. The role of Ctf18 in checkpoint activation is not shared by all Replication Factor C-like complexes, because proteomic analysis revealed that cells lacking Elg1 (the major subunit of a different Replication Factor C-like complex) display a different spectrum of chromatin abnormalities. Identification of Ctf18 as a checkpoint protein highlights the usefulness of chromatin proteomic analysis for understanding the in vivo function of proteins that mediate chromatin transactions.

• Diffley JF
• Quality control in the initiation of eukaryotic DNA replication.
• Philos Trans R Soc Lond B Biol Sci. 2011; 366: 3545-53
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• Origins of DNA replication must be regulated to ensure that the entire genome is replicated precisely once in each cell cycle. In human cells, this requires that tens of thousands of replication origins are activated exactly once per cell cycle. Failure to do so can lead to cell death or genome rearrangements such as those associated with cancer. Systems ensuring efficient initiation of replication, while also providing a robust block to re-initiation, play a crucial role in genome stability. In this review, I will discuss some of the strategies used by cells to ensure once per cell cycle replication and provide a quantitative framework to evaluate the relative importance and efficiency of individual pathways involved in this regulation.

• Jones DR, Prasad AA, Chan PK, Duncker BP
• The Dbf4 motif C zinc finger promotes DNA replication and mediates resistance to genotoxic stress.
• Cell Cycle. 2010; 9: 2018-26
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• The Dbf4/Cdc7 kinase (DDK) plays an essential role in stimulating DNA replication by phosphorylating subunits of the Mcm2-7 helicase complex at origins. This kinase complex is itself phosphorylated and removed from chromatin in a Rad53-dependent manner when an S phase checkpoint is triggered. Comparison of Dbf4 sequence across a variety of eukaryotic species has revealed three conserved regions that have been termed motifs N, M and C. The most highly conserved of the three, motif C, encodes a zinc finger, which are known to mediate protein-protein and protein-DNA interactions. Mutation of conserved motif C cysteines and histidines disrupted the association of Dbf4 with ARS1 origin DNA and Mcm2, but not other known ligands including Cdc7, Rad53 or the origin recognition complex subunit Orc2. Furthermore, these mutations impaired the ability of Dbf4 to phosphorylate Mcm2. Budding yeast strains for which the single genomic DBF4 copy was replaced with these motif C mutant alleles were compromised for entry into and progression through S phase, indicating that the observed weakening of the Mcm2 interaction prevents DDK from efficiently stimulating the initiation of DNA replication. Following initiation, Mcm2-7 migrates with the replication fork. Interestingly, the motif C mutants were sensitive to long-term, but not short-term exposure to the genotoxic agents hydroxyurea and methyl methanesulfonate. These results support a model whereby DDK interaction with Mcm2 is important to stabilize and/or restart replication forks during conditions where a prolonged S-phase checkpoint is triggered.

• Chowdhury A et al.
• The DNA unwinding element binding protein DUE-B interacts with Cdc45 in preinitiation complex formation.
• Mol Cell Biol. 2010; 30: 1495-507
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• Template unwinding during DNA replication initiation requires the loading of the MCM helicase activator Cdc45 at replication origins. We show that Cdc45 interacts with the DNA unwinding element (DUE) binding protein DUE-B and that these proteins localize to the DUEs of active replication origins. DUE-B and Cdc45 are not bound at the inactive c-myc replicator in the absence of a functional DUE or at the recently identified ataxin 10 (ATX10) origin, which is silent before disease-related (ATTCT)(n) repeat length expansion of its DUE sequence, despite the presence of the origin recognition complex (ORC) and MCM proteins at these origins. Addition of a heterologous DUE to the ectopic c-myc origin, or expansion of the ATX10 DUE, leads to origin activation, DUE-B binding, and Cdc45 binding. DUE-B, Cdc45, and topoisomerase IIbeta binding protein 1 (TopBP1) form complexes in cell extracts and when expressed from baculovirus vectors. During replication in Xenopus egg extracts, DUE-B and Cdc45 bind to chromatin with similar kinetics, and DUE-B immunodepletion blocks replication and the loading of Cdc45 and a fraction of TopBP1. The coordinated binding of DUE-B and Cdc45 to origins and the physical interactions of DUE-B, Cdc45, and TopBP1 suggest that complexes of these proteins are necessary for replication initiation.

• Sharma A, Kaur M, Kar A, Ranade SM, Saxena S
• Ultraviolet radiation stress triggers the down-regulation of essential replication factor Mcm10.
• J Biol Chem. 2010; 285: 8352-62
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• We report that upon UV radiation insult, mammalian cells specifically down-regulate Mcm10, a protein essential for the initiation and elongation phases of DNA replication. The levels of a majority of replication factors remain unaffected under this condition, implying that Mcm10 is a key node in the regulation of the replication machinery. High doses of ionizing gamma radiation and exposure to a combination of DNA-damaging chemicals do not decrease Mcm10 protein levels, demonstrating that Mcm10 down-regulation is triggered only by UV-specific damage. The decrease of Mcm10 protein levels is not caused by transcriptional inhibition or cleavage by apoptotic enzymes, but results from degradation by the 26 S proteasome. UV-triggered degradation of Mcm10 requires its linker or C-terminal domain. In addition, Mcm10 down-regulation is not limited to cells from a particular lineage. Therefore, our study reveals a mechanism by which mammalian cells effectively inhibit the replication machinery during stress to prevent it from drifting toward a catastrophic path of genomic instability.

• Bochman ML, Schwacha A
• The Mcm complex: unwinding the mechanism of a replicative helicase.
• Microbiol Mol Biol Rev. 2009; 73: 652-83
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• The Mcm2-7 complex serves as the eukaryotic replicative helicase, the molecular motor that both unwinds duplex DNA and powers fork progression during DNA replication. Consistent with its central role in this process, much prior work has illustrated that Mcm2-7 loading and activation are landmark events in the regulation of DNA replication. Unlike any other hexameric helicase, Mcm2-7 is composed of six unique and essential subunits. Although the unusual oligomeric nature of this complex has long hampered biochemical investigations, recent advances with both the eukaryotic as well as the simpler archaeal Mcm complexes provide mechanistic insight into their function. In contrast to better-studied homohexameric helicases, evidence suggests that the six Mcm2-7 complex ATPase active sites are functionally distinct and are likely specialized to accommodate the regulatory constraints of the eukaryotic process.

• Eichinger CS et al.
• Aberrant DNA polymerase alpha is excluded from the nucleus by defective import and degradation in the nucleus.
• J Biol Chem. 2009; 284: 30604-14
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• DNA polymerase alpha is essential for the onset of eukaryotic DNA replication. Its correct folding and assembly within the nuclear replication pre-initiation complex is crucial for normal cell cycle progression and genome maintenance. Due to a single point mutation in the largest DNA polymerase alpha subunit, p180, the temperature-sensitive mouse cell line tsFT20 exhibits heat-labile DNA polymerase alpha activity and S phase arrest at restrictive temperature. In this study, we show that an aberrant form of endogenous p180 in tsFT20 cells (p180(tsFT20)) is strictly localized in the cytoplasm while its wild-type counterpart enters the nucleus. Time-lapse fluorescence microscopy with enhanced green fluorescent protein-tagged or photoactivatable green fluorescent protein-tagged p180(tsFT20) variants and inhibitor analysis revealed that the exclusion of aberrant p180(tsFT20) from the nucleus is due to two distinct mechanisms: first, the inability of newly synthesized (cytoplasmic) p180(tsFT20) to enter the nucleus and second, proteasome-dependent degradation of nuclear-localized protein. The nuclear import defect seems to result from an impaired association of aberrant de novo synthesized p180(tsFT20) with the second subunit of DNA polymerase alpha, p68. In accordance, we show that RNA interference of p68 results in a decrease of the overall p180 protein level and in a specific increase of cytoplasmic localized p180 in NIH3T3 cells. Taken together, our data suggest two mechanisms that prevent the nuclear expression of aberrant DNA polymerase alpha.

• Warren EM, Huang H, Fanning E, Chazin WJ, Eichman BF
• Physical interactions between Mcm10, DNA, and DNA polymerase alpha.
• J Biol Chem. 2009; 284: 24662-72
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• Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase alpha (pol alpha), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol alpha. However, the mechanism by which Mcm10 interacts with pol alpha on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID*ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286-310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol alpha within the replication fork.

• Eisenberg S, Korza G, Carson J, Liachko I, Tye BK
• Novel DNA binding properties of the Mcm10 protein from Saccharomyces cerevisiae.
• J Biol Chem. 2009; 284: 25412-20
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• The Mcm10 protein is essential for chromosomal DNA replication in eukaryotic cells. We purified the Saccharomyces cerevisiae Mcm10 (ScMcm10) and characterized its DNA binding properties. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that ScMcm10 binds stably to both double strand (ds) DNA and single strand (ss) DNA. On short DNA templates of 25 or 50 bp, surface plasmon resonance analysis showed a approximately 1:1 stoichiometry of ScMcm10 to dsDNA. On longer dsDNA templates, however, multiple copies of ScMcm10 cooperated in the rapid assembly of a large, stable nucleoprotein complex. The amount of protein bound was directly proportional to the length of the DNA, with an average occupancy spacing of 21-24 bp. This tight spacing is consistent with a nucleoprotein structure in which ScMcm10 is aligned along the helical axis of the dsDNA. In contrast, the stoichiometry of ScMcm10 bound to ssDNA of 20-50 nucleotides was approximately 3:1 suggesting that interaction with ssDNA induces the assembly of a multisubunit ScMcm10 complex composed of at least three subunits. The tight packing of ScMcm10 on dsDNA and the assembly of a multisubunit complex on ssDNA suggests that, in addition to protein-DNA, protein-protein interactions may be involved in forming the nucleoprotein complex. We propose that these DNA binding properties have an important role in (i) initiation of DNA replication and (ii) formation and maintenance of a stable replication fork during the elongation phase of chromosomal DNA replication.

• Pai CC, Garcia I, Wang SW, Cotterill S, Macneill SA, Kearsey SE
• GINS inactivation phenotypes reveal two pathways for chromatin association of replicative alpha and epsilon DNA polymerases in fission yeast.
• Mol Biol Cell. 2009; 20: 1213-22
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• The tetrameric GINS complex, consisting of Sld5-Psf1-Psf2-Psf3, plays an essential role in the initiation and elongation steps of eukaryotic DNA replication, although its biochemical function is unclear. Here we investigate the function of GINS in fission yeast, using fusion of Psf1 and Psf2 subunits to a steroid hormone-binding domain (HBD) to make GINS function conditional on the presence of beta-estradiol. We show that inactivation of Psf1-HBD causes a tight but rapidly reversible DNA replication arrest phenotype. Inactivation of Psf2-HBD similarly blocks premeiotic DNA replication and leads to loss of nuclear localization of another GINS subunit, Psf3. Inactivation of GINS has distinct effects on the replication origin association and chromatin binding of two of the replicative DNA polymerases. Inactivation of Psf1 leads to loss of chromatin binding of DNA polymerase epsilon, and Cdc45 is similarly affected. In contrast, chromatin association of the catalytic subunit of DNA polymerase alpha is not affected by defective GINS function. We suggest that GINS functions in a pathway that involves Cdc45 and is necessary for DNA polymerase epsilon chromatin binding, but that a separate pathway sets up the chromatin association of DNA polymerase alpha.

• Park JH, Bang SW, Jeon Y, Kang S, Hwang DS
• Knockdown of human MCM10 exhibits delayed and incomplete chromosome replication.
• Biochem Biophys Res Commun. 2008; 365: 575-82
• Display abstract

• In model organisms, MCM10 is required for forming the pre-initiation complex for initiation of chromosome replication and is involved in the elongation step. To investigate the role of MCM10 in human chromosome replication, we used small interfering RNA (siRNA) in MCM10-knockdown experiments and found that knockdown accumulated S and G2 phase cells. The chromosome replication of MCM10-knockdown cells was slowed during early and mid S phases, although Cdc45, Polalpha, and PCNA proteins were loaded onto the chromatin, and was aberrant during late S phase. Our results indicate that MCM10 is essential for the efficient elongation step of chromosome replication.

• Swaminathan S, Kile AC, MacDonald EM, Koepp DM
• Yra1 is required for S phase entry and affects Dia2 binding to replication origins.
• Mol Cell Biol. 2007; 27: 4674-84
• Display abstract

• The Saccharomyces cerevisiae F-box protein Dia2 is important for DNA replication and genomic stability. Using an affinity approach, we identified Yra1, a transcription-coupled mRNA export protein, as a Dia2 interaction partner. We find that yra1 mutants are sensitive to DIA2 expression levels. Like Dia2, Yra1 associates with chromatin and binds replication origins, suggesting that they may function together in DNA replication. Consistent with this idea, Yra1 and Dia2 coimmunoprecipitate with Hys2, a subunit of DNA polymerase delta. The C terminus of Yra1 is required to interact with Dia2. A yra1 mutant that lacks this domain is temperature sensitive yet has no apparent defect in RNA export. Remarkably, this mutant also fails to enter S phase at the nonpermissive temperature. Significantly, other mutants in transcription-coupled export do not exhibit S phase entry defects or sensitivity to DIA2 expression levels. Together, these results indicate that Yra1 has a role in DNA replication distinct from its role in mRNA export. Furthermore, Dia2 binding to replication origins is significantly reduced when association with Yra1 is compromised, suggesting that one aspect of the role of Yra1 in DNA replication may involve recruiting Dia2 to chromatin.

• Camahort R, Li B, Florens L, Swanson SK, Washburn MP, Gerton JL
• Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore.
• Mol Cell. 2007; 26: 853-65
• Display abstract

• The kinetochore is a complex multiprotein structure located at centromeres that is essential for proper chromosome segregation. Budding-yeast Cse4 is an essential evolutionarily conserved histone H3 variant recruited to the centromere by an unknown mechanism. We have identified Scm3, an inner kinetochore protein that immunopurifies with Cse4. Scm3 is essential for viability and localizes to all centromeres. Construction of a conditional SCM3 allele reveals that depletion results in metaphase arrest, with duplicated spindle poles, short spindles, and unequal DNA distribution. The metaphase arrest is mediated by the mitotic spindle checkpoint being dependent on Mad1 and the Aurora kinase B homolog Ipl1. Scm3 interacts with both Ndc10 and Cse4 and is essential to establish centromeric chromatin after DNA replication. In addition, Scm3 is required to maintain kinetochore function throughout the cell cycle. We propose a model in which Ndc10/Scm3 binds to centromeric DNA, which is in turn essential for targeting Cse4 to centromeres.

• Okorokov AL et al.
• Hexameric ring structure of human MCM10 DNA replication factor.
• EMBO Rep. 2007; 8: 925-30
• Display abstract

• The DNA replication factor minichromosome maintenance 10 (MCM10) is a conserved, abundant nuclear protein crucial for origin firing. During the transition from pre-replicative complexes to pre-initiation complexes, MCM10 recruitment to replication origins is required to provide a physical link between the MCM2-7 complex DNA helicase and DNA polymerases. Here, we report the molecular structure of human MCM10 as determined by electron microscopy and single-particle analysis. The MCM10 molecule is a ring-shaped hexamer with large central and smaller lateral channels and a system of inner chambers. This structure, together with biochemical data, suggests that this important protein uses its architecture to provide a docking module for assembly of the molecular machinery required for eukaryotic DNA replication.

• De Falco M, Ferrari E, De Felice M, Rossi M, Hubscher U, Pisani FM
• The human GINS complex binds to and specifically stimulates human DNA polymerase alpha-primase.
• EMBO Rep. 2007; 8: 99-103
• Display abstract

• The eukaryotic GINS complex has an essential role in the initiation and elongation phases of genome duplication. It is composed of four paralogous subunits--Sld5, Psf1, Psf2 and Psf3--which are ubiquitous and evolutionarily conserved in eukaryotic organisms. Here, we report the biochemical characterization of the human GINS complex (hGINS). The four hGINS subunits were coexpressed in Escherichia coli in a highly soluble form and purified as a complex. hGINS was shown to interact directly with the heterodimeric human DNA primase, by using either surface plasmon resonance measurements or by immunoprecipitation experiments carried out with anti-hGINS antibodies. The DNA polymerase alpha-primase synthetic activity was specifically stimulated by hGINS on various primed DNA templates. The significance of these findings is discussed in view of the molecular dynamics at the human replication fork.

• Semple JW et al.
• An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes.
• EMBO J. 2006; 25: 5150-8
• Display abstract

• The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G(1) phase assembly of pre-replicative complexes (pre-RC). Only the Orc1-5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6-YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre-RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G(1), Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.

• Das-Bradoo S, Ricke RM, Bielinsky AK
• Interaction between PCNA and diubiquitinated Mcm10 is essential for cell growth in budding yeast.
• Mol Cell Biol. 2006; 26: 4806-17
• Display abstract

• The minichromosome maintenance protein 10 (Mcm10) is an evolutionarily conserved factor that is essential for replication initiation and elongation. Mcm10 is part of the eukaryotic replication fork and interacts with a variety of proteins, including the Mcm2-7 helicase and DNA polymerase alpha/primase complexes. A motif search revealed a match to the proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) box in Mcm10. Here, we demonstrate a direct interaction between Mcm10 and PCNA that is alleviated by mutations in conserved residues of the PIP box. Interestingly, only the diubiquitinated form of Mcm10 binds to PCNA. Diubiquitination of Mcm10 is cell cycle regulated; it first appears in late G(1) and persists throughout S phase. During this time, diubiquitinated Mcm10 is associated with chromatin, suggesting a direct role in DNA replication. Surprisingly, a Y245A substitution in the PIP box of Mcm10 that inhibits the interaction with PCNA abolishes cell proliferation. This severe-growth phenotype, which has not been observed for analogous mutations in other PCNA-interacting proteins, is rescued by a compensatory mutation in PCNA that restores interaction with Mcm10-Y245A. Taken together, our results suggest that diubiquitinated Mcm10 interacts with PCNA to facilitate an essential step in DNA elongation.

• Donato JJ, Chung SC, Tye BK
• Genome-wide hierarchy of replication origin usage in Saccharomyces cerevisiae.
• PLoS Genet. 2006; 2: 141-141
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• Replication origins in a genome are inherently different in their base sequence and in their response to temporal and cell cycle regulation signals for DNA replication. To investigate the chromosomal determinants that influence the efficiency of initiation of DNA replication genome-wide, we made use of a reverse strategy originally used for the isolation of replication initiation mutants in Saccharomyces cerevisiae. In yeast, replication origins isolated from chromosomes support the autonomous replication of plasmids. These replication origins, whether in the context of a chromosome or a plasmid, will initiate efficiently in wild-type cells but show a dramatically contrasted efficiency of activation in mutants defective in the early steps of replication initiation. Serial passages of a genomic library of autonomously replicating sequences (ARSs) in such a mutant allowed us to select for constitutively active ARSs. We found a hierarchy of preferential initiation of ARSs that correlates with local transcription patterns. This preferential usage is enhanced in mutants defective in the assembly of the prereplication complex (pre-RC) but not in mutants defective in the activation of the pre-RC. Our findings are consistent with an interference of local transcription with the assembly of the pre-RC at a majority of replication origins.

• Nedelcheva MN et al.
• Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex.
• J Mol Biol. 2005; 347: 509-21
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• The replicative DNA helicases can unwind DNA in the absence of polymerase activity in vitro. In contrast, replicative unwinding is coupled with DNA synthesis in vivo. The temperature-sensitive yeast polymerase alpha/primase mutants cdc17-1, pri2-1 and pri1-m4, which fail to execute the early step of DNA replication, have been used to investigate the interaction between replicative unwinding and DNA synthesis in vivo. We report that some of the plasmid molecules in these mutant strains became extensively negatively supercoiled when DNA synthesis is prevented. In contrast, additional negative supercoiling was not detected during formation of DNA initiation complex or hydroxyurea replication fork arrest. Together, these results indicate that the extensive negative supercoiling of DNA is a result of replicative unwinding, which is not followed by DNA synthesis. The limited number of unwound plasmid molecules and synthetic lethality of polymerase alpha or primase with checkpoint mutants suggest a checkpoint regulation of the replicative unwinding. In concordance with this suggestion, we found that the Tof1/Csm3/Mrc1 checkpoint complex interacts directly with the MCM helicase during both replication fork progression and when the replication fork is stalled.

• Zhou Y, Wang TS
• A coordinated temporal interplay of nucleosome reorganization factor, sister chromatin cohesion factor, and DNA polymerase alpha facilitates DNA replication.
• Mol Cell Biol. 2004; 24: 9568-79
• Display abstract

• DNA replication depends critically upon chromatin structure. Little is known about how the replication complex overcomes the nucleosome packages in chromatin during DNA replication. To address this question, we investigate factors that interact in vivo with the principal initiation DNA polymerase, DNA polymerase alpha (Polalpha). The catalytic subunit of budding yeast Polalpha (Pol1p) has been shown to associate in vitro with the Spt16p-Pob3p complex, a component of the nucleosome reorganization system required for both replication and transcription, and with a sister chromatid cohesion factor, Ctf4p. Here, we show that an N-terminal region of Polalpha (Pol1p) that is evolutionarily conserved among different species interacts with Spt16p-Pob3p and Ctf4p in vivo. A mutation in a glycine residue in this N-terminal region of POL1 compromises the ability of Pol1p to associate with Spt16p and alters the temporal ordered association of Ctf4p with Pol1p. The compromised association between the chromatin-reorganizing factor Spt16p and the initiating DNA polymerase Pol1p delays the Pol1p assembling onto and disassembling from the late-replicating origins and causes a slowdown of S-phase progression. Our results thus suggest that a coordinated temporal and spatial interplay between the conserved N-terminal region of the Polalpha protein and factors that are involved in reorganization of nucleosomes and promoting establishment of sister chromatin cohesion is required to facilitate S-phase progression.

• Takayama Y, Kamimura Y, Okawa M, Muramatsu S, Sugino A, Araki H
• GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast.
• Genes Dev. 2003; 17: 1153-65
• Display abstract

• Eukaryotic chromosomal DNA replication requires a two-step assembly of replication proteins on origins; formation of the prereplicative complex (pre-RC) in late M and G1 phases of the cell cycle, and assembly of other replication proteins in S phase to load DNA polymerases to initiate DNA synthesis. In budding yeast, assembly of Dpb11 and the Sld3-Cdc45 complex on the pre-RC at origins is required for loading DNA polymerases. Here we describe a novel replication complex, GINS (Go, Ichi, Nii, and San; five, one, two, and three in Japanese), in budding yeast, consisting of Sld5, Psf1 (partner of Sld five 1), Psf2, and Psf3 proteins, all of which are highly conserved in eukaryotic cells. Since the conditional mutations of Sld5 and Psf1 confer defect of DNA replication under nonpermissive conditions, GINS is suggested to function for chromosomal DNA replication. Consistently, in S phase, GINS associates first with replication origins and then with neighboring sequences. Without GINS, neither Dpb11 nor Cdc45 associates properly with chromatin DNA. Conversely, without Dpb11 or Sld3, GINS does not associate with origins. Moreover, genetic and two-hybrid interactions suggest that GINS interacts with Sld3 and Dpb11. Therefore, Dpb11, Sld3, Cdc45, and GINS assemble in a mutually dependent manner on replication origins to initiate DNA synthesis.

• Tanaka S, Diffley JF
• Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase.
• Nat Cell Biol. 2002; 4: 198-207
• Display abstract

• Cdt1 is essential for loading Mcm2-7 proteins into prereplicative complexes (pre-RCs) during replication licensing and has been found in organisms as diverse as fission yeast and humans. We have identified a homologue of Cdt1 in Saccharomyces cerevisiae, which is required for pre-RC assembly. We show that, like Mcm2-7p, Cdt1p accumulates in the nucleus during G1 phase and is excluded from the nucleus later in the cell cycle by cyclin dependent kinases (cdks). Cdt1p interacts with the Mcm2--7p complex, and the nuclear accumulation of these proteins during G1 is interdependent. This coregulation of Cdt1p and Mcm2-7p represents a novel level of pre-RC control.

• Schepers A, Diffley JF
• Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein.
• J Mol Biol. 2001; 308: 597-608
• Display abstract

• The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.

• Lei M, Tye BK
• Initiating DNA synthesis: from recruiting to activating the MCM complex.
• J Cell Sci. 2001; 114: 1447-54
• Display abstract

• The exact duplication of a genome once per cell division is required of every proliferating cell. To achieve this goal, eukaryotes adopt a strategy that limits every replication origin to a single initiation event within a narrow window of the cell cycle by temporally separating the assembly of the pre-replication complex (pre-RC) from the initiation of DNA synthesis. A key component of the pre-RC is the hexameric MCM complex, which is also the presumed helicase of the growing forks. An elaborate mechanism recruits the MCM complex to replication origins, and a regulatory chain reaction converts the poised, but inactive, MCM complex into an enzymatically active helicase. A growing list of proteins, including Mcm10 and Cdt1, are involved in the recruitment process. Two protein kinases, the Cdc7-Dbf4 kinase (DDK) and the cyclin-dependent kinase (CDK), trigger a chain reaction that results in the phosphorylation of the MCM complex and finally in the initiation of DNA synthesis. A composite picture from recent studies suggests that DDK is recruited to the pre-RC during G1 phase but must wait until S phase to phosphorylate the MCM complex. CDK is required for the recruitment of Cdc45 and other downstream components of the elongation machinery.

• Labib K, Kearsey SE, Diffley JF
• MCM2-7 proteins are essential components of prereplicative complexes that accumulate cooperatively in the nucleus during G1-phase and are required to establish, but not maintain, the S-phase checkpoint.
• Mol Biol Cell. 2001; 12: 3658-67
• Display abstract

• A prereplicative complex (pre-RC) of proteins is assembled at budding yeast origins of DNA replication during the G1-phase of the cell cycle, as shown by genomic footprinting. The proteins responsible for this prereplicative footprint have yet to be identified but are likely to be involved in the earliest stages of the initiation step of chromosome replication. Here we show that MCM2-7 proteins are essential for both the formation and maintenance of the pre-RC footprint at the origin ARS305. It is likely that pre-RCs contain heteromeric complexes of MCM2-7 proteins, since degradation of Mcm2, 3, 6, or 7 during G1-phase, after pre-RC formation, causes loss of Mcm4 from the nucleus. It has been suggested that pre-RCs on unreplicated chromatin may generate a checkpoint signal that inhibits premature mitosis during S-phase. We show that, although mitosis does indeed occur in the absence of replication if MCM proteins are degraded during G1-phase, anaphase is prevented if MCMs are degraded during S-phase. Our data indicate that pre-RCs do not play a direct role in checkpoint control during chromosome replication.

• Kawasaki Y, Hiraga S, Sugino A
• Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae.
• Genes Cells. 2000; 5: 975-89
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• BACKGROUND: MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2-7 protein complex that may be functioning as the replication-licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks. RESULTS: We show that an mcm10 mutation causes a slow progression of DNA synthesis and a loss of chromosome integrity during the S phase and prevents entry into mitosis, despite apparent completion of chromosomal DNA replication at nonpermissive temperatures. Furthermore, Mcm10p interacts genetically with the origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerases epsilon and delta. Mcm10p is an abundant protein (approximately 4 x 10(4) copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fractions during all phases of the cell cycle. When it is visualized by the chromosome-spreading method followed by immunostaining, Mcm10p forms punctate foci on chromatin throughout the cell cycle and these foci mostly overlap with those of Orc1p, a component of ORC. CONCLUSIONS: These results suggest that Mcm10p, like the Mcm2-7 proteins, is a critical component of the prereplication chromatin and acts together with ORC during the initiation of chromosomal DNA replication; in addition, Mcm10p plays an important role during the elongation of DNA replication.

• Izumi M et al.
• The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase.
• Nucleic Acids Res. 2000; 28: 4769-77
• Display abstract

• Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

• Walter J, Newport J
• Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha.
• Mol Cell. 2000; 5: 617-27
• Display abstract

• We report that a plasmid replicating in Xenopus egg extracts becomes negatively supercoiled during replication initiation. Supercoiling requires the initiation factor Cdc45, as well as the single-stranded DNA-binding protein RPA, and therefore likely represents origin unwinding. When unwinding is prevented, Cdc45 binds to chromatin whereas DNA polymerase alpha does not, indicating that Cdc45, RPA, and DNA polymerase alpha bind chromatin sequentially at the G1/S transition. Whereas the extent of origin unwinding is normally limited, it increases dramatically when DNA polymerase alpha is inhibited, indicating that the helicase that unwinds DNA during initiation can become uncoupled from the replication fork. We discuss the implications of these results for the location of replication start sites relative to the prereplication complex.

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